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DISSERTAO DE MESTRADO TOXICOLOGIA E CONTAMINAO AMBIENTAIS

Anticancer activity against glioblastoma

cell lines by compounds present in algae,

alone and in combination with anticancer

drugs

Joana Rodrigues Neves Ferreira

M 2016

JOANA RODRIGUES NEVES FERREIRA

Anticancer activity against glioblastoma cell lines by compounds

present in algae, alone and in combination with anticancer drugs

Dissertation for the degree of Master in

Environmental Contamination and Toxicology, presented to

Institute of Biomedical Sciences Abel Salazar

of the University of Porto

Supervisor Alice Fernanda Abreu Ramos

Category Post-Doc Researcher

Affiliation Interdisciplinary Centre of Marine and

Environmental Research, University of Porto

Co-supervisor Eduardo Jorge Sousa da Rocha

Category Full Professor

Affiliation Institute of Biomedical Sciences Abel Salazar,

University of Porto

i

Acknowledgements

I would like to thank the succeeding people:

Doctor Alice Ramos, my advisor, for all the support, advice, cooperation and friendship that

you provided me. This support during this period of my academic life was very important

and appreciated, and definitely not forgotten.

Professor Eduardo Rocha, my co-advisor, for giving me a chance to work on this project

and for all the support and management provided during this time.

Doctor Amaya Azqueta, that was so kind to receive me in her laboratory in Pamplona,

Spain, and for helping me and guide me with any questions that I had.

Doctor Violeta Gmez, and all laboratory colleagues at CIMA, Spain, for making me fill very

welcome and integrated as well for providing a very good guidance and support.

Tnia Almeida, my best friend, for her priceless assistance in the practical work and for

keeping me motivated and helping me during the writing stage. Without her, this thesis

would be very difficult!

To all laboratory colleagues at PATH group, especially to Mariana Abreu, because they

were very supportive and helpful at all times.

Finally, and more especially, I would like to thank my parents and my brother for all the

encouragement and the opportunity to make this possible.

This research was partially financed by the Research Line NOVELMAR Novel Marine

Products with Biotechnological Applications, integrated into the Structured Program of

R&D&I INNOVMAR - Innovation and Sustainability in the Management and Exploitation of

Marine Resources (reference NORTE-01-0145-FEDER-000035), funded by the Northern

Regional Operational Programme (NORTE2020) through the European Regional

Development Fund (ERDF).

iii

Abstract

Glioblastomas (GBM) are the most aggressive and lethal tumor of the central nervous

system with an average life expectancy of only 1 to 2 years after diagnosis, even with the

use of standard treatment of surgery, radiation, and chemotherapy. There are several

anticancer drugs with alkylating properties that have been used in the therapy of malignant

gliomas. Temozolomide (TMZ) is an anticancer drug widely used, sometimes in

combination with ionizing radiation. However, one of the downsides of using this type of

drugs in the treatment of GBM is the development of cancer drug resistance. As so,

research of bioactive compounds with anticancer activity has been, in the last decades,

heavily explored in terrestrial environments. However, also marine environments appear as

a promising source of new bioactive compounds. Several natural compounds, existing in

seaweed, have shown cytotoxic activity in in vitro and in vivo models acting through different

molecular mechanisms, such as antioxidant, antiproliferative, DNA damage/DNA repair and

apoptosis induction. Less explored is the ability of seaweed compounds to enhance the

cytotoxic action of anticancer drugs. So, the combination of natural compounds with

conventional drugs can be seen as an interesting strategy to be studied.

The aim of this work was to assess the anticancer effect of fucoxanthin (Fx) and

phloroglucinol (Ph), two natural compounds present in many seaweed, either alone or in

combination with TMZ, in two glioblastoma cell lines (U251 and T98G). Results showed that

both cell lines are clearly different in terms of sensitivity to TMZ, in that U251 demonstrated

a higher sensitivity then T98G cells. Also, Fx and Ph demonstrated cytotoxic and anti-

proliferative effects against both cell lines, being the effects more evident in U251 cells.

However, the mechanisms behind these effects do not appear to be the induction of cell

death by apoptosis or DNA damage. The combination demonstrating the highest interest is

10 M TMZ + 10 M Fx, in U251 cells, in which was seen that Fx potentiates the TMZ

action. Nevertheless, more studies are required to elucidate the mechanisms involved. In

conclusion, Fx and Ph showed antiproliferative effects against glioblastoma cell lines and

in the case of Fx may potentiate the activity of the alkylating drug TMZ. Fx and Ph appear

as promising anti-glioblastoma agents that should be more explored.

v

Resumo

Glioblastomas (GBM) so dos tumores mais agressivos e letais do sistema nervoso

central com uma esperana mdia de vida de 1 a 2 anos aps diagnstico, mesmo com o

uso de tratamentos envolvendo cirurgia, radioterapia e quimioterapia. Existem diversos

frmacos anticancergenos com propriedades alquilantes que tm sido utilizados na terapia

de gliomas malignos. A temozolomida (TMZ) um frmaco anticancergeno amplamente

utilizado, por vezes em combinao com a radiao ionizante. No entanto, uma das

desvantagens do uso deste tipo de frmacos no tratamento de glioblastomas o

desenvolvimento de resistncia ao frmaco. Como tal, a procura de novos compostos

bioativos com atividade anticancergena tem sido, nas ltimas dcadas, fortemente

explorada em ambientes terrestres. Contudo, tambm os ambientes marinhos parecem ser

uma fonte promissora de novos compostos bioativos. Vrios compostos naturais,

existentes em algas, tm demonstrado ter atividade citotxica em modelos in vitro e in vivo

atuando atravs de diferentes mecanismos moleculares, atravs de efeitos antioxidantes,

antiproliferativos, indutores de apoptose e de danos/reparao de ADN. Menos explorado

tem sido a capacidade dos compostos extrados de algas de aumentar a ao citotxica

dos frmacos anticancergenos. Deste modo a combinao dos compostos naturais com

os frmacos convencionais poder ser uma estratgia interessante a ser estudada.

O objetivo deste trabalho foi a avaliao dos efeitos anticancergenos da fucoxantina

(Fx) e do floroglucinol (Ph), dois compostos naturais que esto presentes em algas, quer

sozinhos ou em combinao com a TMZ, em duas linhagens celulares de glioblastoma

(U251 e T98G). Os resultados mostraram, claramente, que as duas linhagens diferem em

termos de sensibilidade TMZ, em que as clulas U251 demonstraram ser mais sensveis

do que as clulas T98G. Alm disso, a Fx e o Ph demonstraram efeitos citotxicos e anti

proliferativos em ambas as linhagens, sendo estes efeitos mais evidentes nas clulas

U251. No entanto, os mecanismos responsveis por estes efeitos no parecem estar

relacionados com a induo de morte celular por apoptose ou por danos no ADN. A

combinao que mostrou ser de maior interesse a 10 M TMZ + 10 M Fx, nas clulas

U251; situao em que a Fx potenciou a ao da TMZ. Apesar disto, mais estudos so

necessrios para ajudar a elucidar os mecanismos envolvidos. Em concluso, a Fx e o Ph

demonstraram efeitos antiproliferativos contra linhagens celulares de glioblastoma e no

caso da Fx poder potenciar a atividade do frmaco alquilante TMZ. A Fx e o Ph parecem

ser agentes anticancergenos promissores que devem ser mais explorados.

vii

Index

Acknowledgements .......................................................................................................... i

Abstract ........................................................................................................................... iii

Resumo ............................................................................................................................ v

Abbreviations .................................................................................................................. ix

Chapter 1 General Introduction .................................................................................. 1

1. Cancer ................................................................................................................... 3

1.1. Carcinogenesis ................................................................................................. 3

1.2. Glioblastomas ................................................................................................... 4

1.2.1. Molecular mechanisms involved .............................................................. 5

1.2.2. Treatment Chemotherapy ...................................................................... 5

1.2.3. Main me